In preliminary experiments, I have identified a previously unrecognized protease inhibitor in human plasma. Initial studies indicate that this inhibitor is a protein with an apparent molecular weight of 72,000, that it inactivates thrombin by forming a very stable 1:1 stoichiometric complex with the protease, and that it is immunologically distinct from all of the known plasma protease inhibitors. A striking property of the new inhibitor is that its action is markedly accelerated by heparin. In addition, at relatively high concentrations of heparin, this inhibitor competes quite effectively for thrombin with antithrombin III, although antithrombin III has previously been considered to be the only quantitatively significant inhibitor of thrombin in plasma. In this application, I propose to do the following: 1) Purify the protein to hemogeneity. 2) Develop a specific immunoassay for the new inhibitor in plasma. 3) Determine the amino acid and carbohydrate compositions, partial NH2- and COOH-terminal amino acid sequences, molecular weight, sedimentation coefficient, and extinction co-efficient of the inhibitor. 4) Determine the ability of the purified inhibitor to inactivate coagulation factors IIa, Xa, VIIa, IXa, XIa, XIIa (fragments), kallekrein, plasmin, and activated protein C. 5) Establish the heparin-dependence and stoichiometry of inhibition of the proteases in (4). 6) Determine the 2nd-order rate constant and/or the equilibrium dissociation constant of the inhibitor-protease complex in the presence and absence of heparin. 7) Fractionate heparin by size and by affinity of binding to the inhibitor, and compare the ability of each fraction to activate both the new inhibitor and antithrombin III. 8) Determine the dissociation constant and stoichiometry of binding of heparin to the new inhibitor. 9) Determine whether the new inhibitor undergoes limited proteolysis during complex formation with a protease. 10) Isolate and sequence the proteolytic fragments, if found, in (9) to investigate the structure of the reactive site of the inhibitor.